Porcine Placenta Hydrolysates Enhance Osteoblast Differentiation Through Their Antioxidant Activity and Effects on ER Stress

Lee et al. BMC Complementary and Alternative Medicine (2016) 16:291 DOI 10.1186/s12906-016-1274-y

RESEARCH ARTICLE Open Access Porcine placenta hydrolysates enhance osteoblast differentiation through their antioxidant activity and effects on ER stress Hwa-Young Lee1, Han-Jung Chae1, Sun-Young Park2 and Jong-Hyun Kim3*

Abstract Background: Osteoporosis is a disease characterized by decreased bone strength, decreased bone mass, and bone deterioration. Oxidative damage is an important contributor to functional changes in the development of osteoporosis. Here we found that porcine placenta hydrolysates (PPHs) protect MC3T3-E1 osteoblastic cells against hydrogen peroxide (H2O2)-induced oxidative damage. Methods: In vitro cell viability was determined using trypan blue dye exclusion. ER stress and apoptosis were evaluated using immunoblotting and a commercially available caspase kit. ALP, osteocalcin, Runx2, and osterix expression levels were evaluated by RT-PCR using isolated RNA. ROS, NADPH oxidase, and SOD activity levels were also measured.

Results: We investigated the mechanisms underlying PPH-mediated inhibition of H2O2-induced ER stress and ROS production. PPHs also regulated osteoblast differentiation via the upregulation of alkaline phosphatase (ALP) expression in MC3T3-E1 osteoblastic cells. Also, treatment with PPHs enhanced the transcription of osteocalcin, Runx2, and osterix. These effects were all associated with the antioxidant actions of PPHs. Moreover, PPHs reversed the decrease in SOD activity, decreased ROS release, and inhibited NADPH oxidase activity in H2O2-treated MC3T3-E1 osteoblastic cells.

Conclusions: PPHs protect cells against H2O2-induced cell damage when ER stress is involved. In addition, PPHs enhance osteoblast differentiation. This enhancement likely explains the regulatory effect of PPHs on bone metabolism disturbances, i.e. PPHs control ER stress and the related ROS production in osteoblasts. Keywords: Osteoporosis, Oxidative damage, Alkaline phosphatase, Apoptosis, ROS

Abbreviations: ALP, Alkaline Phosphatase; ER, Endoplasmic Reticulum; H2O2, Hydrogen Peroxide; PPHs, Porcine Placenta Hydrolysates; ROS, Reactive Oxygen Species; Tg, Thapsigargin

Background pathological conditions. Osteoblasts are secretory cells Osteoporosis is characterized by decreased bone strength, with well-developed endoplasmic reticulum (ER) cristae, decreased bone mass, and bone tissue deterioration. An in which many secretory proteins are folded and then imbalance between bone resorption and bone formation is secreted. The balance of osteoblasts and osteoclastic cells the dominant mechanism causing osteoporosis [1, 2]. is carefully controlled to maintain bone and the related Since new bone formation primarily depends on osteo- endocrinology homeostasis [1, 3, 4]. ROS are one of the blasts, factors that disturb their bone-forming characte- key factors responsible for dysregulation of bone mainten- ristics can lead to bone formation defects or related ance, especially osteoblast physiology. Osteoblasts are coated with high levels of secreted proteins. When these cells do not work effectively, ROS affect the secretory * Correspondence: [email protected] 3Department of Obstetrics and Gynecology, Institute for Medical Sciences, function of the osteoblasts, resulting in detrimental signal- Chonbuk National University Medical School, Jeonju 560-182, Republic of ing to osteoclasts. Furthermore, during severe pathologic Korea stress, osteoblasts undergo apoptosis. This also disturbs Full list of author information is available at the end of the article

© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lee et al. BMC Complementary and Alternative Medicine (2016) 16:291 Page 2 of 10

the balance between osteoblasts and osteoclasts and Extraction and quantitation of PPHs using HPLC-DAD ultimately leads to bone resorption and related disease PPHs were purchased from Codebio Inc. (Cheonan, conditions [5]. Republic of Korea). Placentas were thawed using a The ER plays a major role in controlling protein fold- defroster and then washed with saline at 16 °C to re- ing and secretion in cells. Various acute and chronic move blood and cords. Placentas were hydrolyzed using conditions, including protein misfolding and Ca2+ dis- papain, bromelain, pronase, and Alcalase at 70 ± 0.1 °C turbances, can alter ER function and lead to ER stress for 2 days (pH 4.5–6.0). Next, hydrolytic enzymes were [6–8]. Furthermore, ER stress has been reported to con- inactivated at 100 ± 0.1 °C for 30 min and the resultant tribute to several diseases, including many bone diseases hydrolysates were filtered. The lipids were then removed [9, 10]. Osteoblast apoptosis associated with ER stress is by mixing with calcium and phosphate salts and by filter- one of the predominant mechanisms of osteoporosis ing the insoluble particulate matter. Finally, the hydroly- pathogenesis [11–14]. In stressed osteoblasts, endocrine sates were adjusted to pH 7.0 ± 0.2 using calcium and function, including the production of bone formation phosphate salts. Hydrolysates were mixed with AccQ- hormones (e.g., osteopontin and osteocalcin), is impaired Fluor buffer and Acc-Fluor reagent (Waters Corporation, [15, 16]. Under stress conditions, ROS (e.g. H2O2) have Milford, MA, USA), after which the mixture was heated at been identified as key detrimental messengers. More- 80 °C. An aliquot of the sample was filtered and injected over, ROS have also been associated with ER stress in into an AccQ-Tag column (150 × 2.1 mm, 3 μmparticle other pathological conditions [17–19]. Numerous size; Waters Corporation, Milford, MA, USA) in an HPLC studies have evaluated the role of ROS and ER stress instrument for chromatographic separation. A gradient in bone development [3, 20–22]. Therefore, the iden- mixture of AccQ-Tag Eluent (A) and acetonitrile (B) was tification of interventions that can control ROS-associated used as the mobile phase at a flow rate of 1 mL/min at ER stress is important for preventing and treating bone 30 °C. The initial eluent was 100 % A, which linearly de- disease. creased to 67 % A over 33 min. The eluent was switched The placenta is an organ found exclusively in women to 100 % B for 3 min and then switched back to 100 % A during pregnancy that supplies nutrients and oxygen to for 26 min. Amino acids were detected using a fluo- the developing fetus. The nutritional substances and rescence detector at excitation and emission wavelengths vitamins therein can be extracted in the form of porcine of 250 and 395 nm, respectively. placenta hydrolysates (PPHs). PPHs contain beneficial bioactive components that inhibit aging, sunburns, and Cell culture and viability analysis oxidation [23, 24]. PPHs have also been used for wound The murine calvaria-derived MC3T3-E1 osteoblast-like healing in Korean folk medicine [25, 26] and have been cell line (4 to 10 passages) was purchased from the demonstrated to exert immunomodulatory effects [27, American Type Culture Collection (ATCC; Manassas, 28]. However, the effect of PPHs on bone formation VA, USA). Cells were seeded at 1 × 105 cells/mL and remains unclear. To determine the role of PPHs in maintained in minimum essential medium (α-MEM) the endocrine system, it is important to determine supplemented with 10 % fetal bovine serum (FBS) and the effects of PPHs on osteoblasts, a representative penicillin-streptomycin (Invitrogen). Cells were incu- endocrine cell associated with stress conditions. The bated at 37 °C in a 95 % air/5 % CO2 atm. Cell viability aim of our study was to evaluate the effects of PPHs was detected using a trypan blue exclusion assay. Briefly, 4 on H2O2-induced oxidative damage and the related MC3T3-E1 cells (5 × 10 cells/well) were incubated in ER stress in MC3T3-E1 osteoblastic cells. We found 12-well plates overnight and treated for 24 h with drugs that PPHs regulate oxidative ER stress and the associ- in medium containing 10 % serum. Cells were washed ated activation of antioxidants, thus enhancing differen- with sterile phosphate-buffered saline (PBS), treated with tiation of MC3T3-E1 osteoblastic cells in the presence 0.25 % trypsin-EDTA (GIBCO BRL), and harvested. of H2O2. Cells were diluted in 0.1 % trypan blue (GIBCO BRL) and then counted under a light microscope. Methods Materials Immunoblotting PPHs were purchased from Codebio Inc. (Cheonan, For immunoblotting, MC3T3-E1 osteoblastic cells were Republic of Korea). Hydrogen peroxide and thapsigargin lysed with extraction buffer. Proteins in the resultant were obtained from Sigma Chemical Company (St. lysates (40 μg) were resolved on a polyacrylamide gel Louis, MO, USA). Caspase-3 and -12 activity kits were and transferred to a nitrocellulose membrane. The blots obtained from BioVision (Mountain View, CA, USA). were probed overnight at 4 °C with primary antibodies, All other reagents used were of analytical grade and washed, and probed again with species-specific sec- were generally obtained from Sigma. ondary antibodies coupled to horseradish peroxidase Lee et al. BMC Complementary and Alternative Medicine (2016) 16:291 Page 3 of 10

(GE Healthcare, Piscataway, NJ, USA). Chemilumines- forward primer: 5′-ATCACCATCTTCCAGGAG-3′, re- cence reagents (GE Healthcare) were used for detection. verse primer: 5′-ATGGACTGTGGTCATGAG-3′. Re- Primary antibodies consisted of rabbit anti-GADD153/C/ verse transcription was performed by incubating the EBP homologous protein (CHOP), rabbit-anti-PERK, rat reactions at 37 °C for 15 min and then at 85 °C for 5 s. anti-GRP78, rabbit anti-ATF6α, mouse anti-eIF2α, mouse For polymerase chain reaction amplification, an initial anti-β-actin (Santa Cruz Biotechnologies, Inc., Santa Cruz, denaturation step was performed at 94 °C for 3 min, CA, USA), rabbit anti-IRE1α, and rabbit anti-p-eIF2 (Cell followed by annealing at 55 °C for 20 s and elongation at Signaling Technologies, Inc., Danvers, MA, USA). 72 °C for 45 s. In total, 35 cycles were performed.

Measurement of caspase-3 activity NADPH oxidase activity assay To analyze caspase-3 activity, pellets were resuspended Cells were seeded in six-well plates and cultured for in extraction buffer [25 mM HEPES (pH 7.4), 0.1 % 48 h. Next, the cells were treated with 100 μMH2O2 for Triton X-l00, 10 % glycerol, 5 mM DTT] and spun by 6 h in the presence or absence of 100 μg/mL PPHs. centrifugation at 13,000 rpm at 4 °C for 30 min. Soluble NADPH oxidase activity was determined based on protein (40 μg) was mixed with 100 μM caspase-3- superoxide-induced lucigenin photoemission as described specific substrate Ac-DEVD-AFC (Sigma-Aldrich) and by Rao and Maddala et al. [29]. Enzymatic assays were incubated at 37 °C. Caspase-3 activity was analyzed by performed in a final volume of 0.2 ml containing 50 mM monitoring fluorogenic AFC release at 37 °C. Substrate phosphate buffer (pH 7.0), 1 mM EGTA, 150 mM sucrose, cleavage was monitored at 405 nm using a SPECTRAmax 0.5 mM lucigenin, 0.1 mM NADPH, and cell lysis solu- 340 microplate reader and analyzed using SOFTmax PRO tion. Enzymatic reactions were initiated by the addition of software (Molecular Devices, Sunnyvale, CA, USA). lucigenin. Photoemission, expressed as relative light units, was measured every minute for 10 min using a lumin- Measurement of caspase-12 activity ometer. Assays were performed in the dark at room To analyze caspase-12 activity, pellets were measured by temperature with all appropriate controls. detecting free AFC cleavage by caspase-12-specific sub- strates. These experiments were performed with a Superoxide dismutase (SOD) activity assay caspase-12 Assay Kit (Biovision, San Francisco, CA, Cells were seeded in six-well plates and cultured for USA). After the lysates were incubated with ATAD-AFC 48 h. The cells were then treated with 100 μMH2O2 for for 2 h at 37 °C, the absorbance of each sample was ana- 6 h in the presence or absence of 100 μg/mL PPHs. lyzed at 505 nm. Next, cells were harvested and the level of SOD activity was determined using a SOD assay kit (k335-100, Biovi- Measurement of alkaline phosphatase activity sion) according to the manufacturer’s instructions. To analyze ALP activity, cells were treated with Krebs (control), PPHs (100 μg/mL), H2O2 (400 μM), and in the DCFDA assay (ROS production) presence or absence of 100 μg/mL PPHs. After treat- The cellular ROS level was measured by following the ment for 4 h, cells were lysed. The amount of ALP activ- protocol described by Badham et al. [30]. Briefly, cells ity and the protein concentration were measured in each were treated with 100 μMH2O2 at 37 °C in the presence supernatant using an ALP activity assay kit (Cell Biolabs, or absence of 100 μg/mL PPHs for 6 h. Next, cells were San Diego, CA, USA). incubated with 10 μM2′,7′-dichlorofluorescein diace- tate (DCFDA) at 37 °C for an additional 30 min. The RT-PCR fluorescence intensity of 2′,7′-dichlorofluorescein, a The mRNA levels of ALP, osteocalcin, Runx2, osterix, product of the reaction between DCFDA and cellular and GAPDH were determined using a PrimeScript™ RT ROS, was analyzed using a fluorescence reader (Spectra- reagent Kit (TaKaRa Bioscience, Kyoto, Japan). The se- Max 190, Molecular Devices, Sunnyvale, CA, USA). Data quences of the primers used for RT-PCR were as follows: were normalized to the H2O2 absorbance values. ALP, forward primer: 5′-CCATGGTAGATTACGCTC ACA-3′, reverse primer: 5′-ATGGAGGATTCCAGATA Statistical analysis CAGG-3′; osteocalcin, forward primer: 5′-AGCTAT Results are presented as means ± standard errors of CAGACCAGTATGGCT-3′, reverse primer: 5′-TTTTG the mean (SEMs) for multiple wells tested in at least GAGCTGCTGTGACATC-3′; Runx2, forward primer: three separate experiments. MicroCal Origin software 5′-CTCAGTGATTTAGGGCGCATT-3′, reverse primer: (Northampton, MA, USA) was used for statistical calcula- 5′-AGGGGTAAGACTGGTCATAGG-3′; Osterix, for- tions. Differences were tested for significance using one- ward primer: 5′- CGGGTCAGGTACAGTG-3′, reverse way analysis of variance (ANOVA) with Duncan’s multiple primer: 5′- ACCATGACGACAAGGG-3′; and GAPDH, range test. Lee et al. BMC Complementary and Alternative Medicine (2016) 16:291 Page 4 of 10

2+ Results H2O2 for 6 h or 0.1 μM Tg (thapsigargin, a Ca -ATPase PPHs protect against H2O2-induced cell death in inhibitor) for 24 h to induce ER stress. To investigate MC3T3-E1 osteoblastic cells the ER stress response, the levels of GRP78, CHOP, p- We first determined if PPHs exert concentration- PERK, p-eIF2α, p-IRE1-α, and ATF6α were analyzed by dependent effects on cell viability. Treatment with PPHs immunoblotting. The levels of these proteins were all for 6 h at concentrations ranging from 25 to 100 μg/mL significantly increased in cells treated with 400 μM did not have any significant effect on cell survival H2O2 or 0.1 μM Tg. However, treatment with PPHs (Fig. 1a). However, treatment of cells for 6 h with 100, inhibited this upregulation of GRP78, CHOP p-PERK, p- 200, or 400 μMH2O2 markedly increased cell death in a eIF2α, p-IRE1-α, and ATF6α (Fig. 2a and b). Moreover, concentration-dependent manner (Fig. 1b). Interestingly, PPHs inhibited H2O2-induced cell injury. Apoptosis treatment of cells with 400 μMH2O2 and 25, 50, or levels were measured by flow cytometry and are ex- 100 μg/mL PPHs markedly affected cell survival in a pressed in units of mean fluorescence intensity. As concentration- and time-dependent manner (Fig. 1c and shown in Fig. 3a-b, PPHs protected osteoblasts against d). Moreover, treatment with 100 μg/mL PPHs for H2O2-induced apoptosis. H2O2 treatment also induced 30 min significantly protected cells against H2O2-in- nuclear condensation and fragmentation, characteristics duced (400 μM) cell damage. These findings suggest that of apoptosis. Pretreatment with PPHs markedly at- PPHs significantly protect MC3T3-E1 osteoblastic cells. tenuated these effects (Fig. 3a). Moreover, the apoptosis level was 31.55 ± 1.02 % after H2O2 treatment for 6 h, PPHs protect against H2O2-induced apoptosis in MC3T3-E1 whereas the apoptosis level in the PPHs group was osteoblastic cells 15.33 ± 2.1 % (Fig. 3b). These results confirm the pro- To evaluate whether PPHs could protect against ER tective effects of PPHs against H2O2-induced apoptosis stress in osteoblasts, cells were treated with 400 μM in cells. Caspase-12 activation is associated with

Fig. 1 PPHs regulate H2O2-induced cell death in MC3T3-E1 osteoblastic cells. a To test the cell viability of PPHs in MC3T3-E1 cells, cells were treated with 0, 25, 50, or 100 μg/mL PPHs only for 6 h. Cell viability was estimated via the trypan blue exclusion test. Untreated cells were used as the control.

b To test the cell viability of H2O2 in MC3T3-E1 cells, cells were treated with 0, 100, 200, or 400 μMH2O2 only for 6 h. Cell viability was estimated via the trypan blue exclusion test. Untreated cells were used as the control. c Cells were treated with 0, 25, 50, or 100 μg/mL PPHs in the presence or

absence of 400 μMH2O2 for 6 h, and cell survival was assessed. d Cells were exposed to 400 μMH2O2 in the presence or absence of 100 μg/mL PPHs * for 0, 2, 4, or 6 h, after which cell viability was assessed. p < 0.05 versus cells treated with H2O2 alone (n =3).PPHs porcine placenta hydrolysates Lee et al. BMC Complementary and Alternative Medicine (2016) 16:291 Page 5 of 10

Fig. 2 PPHs decrease the H2O2-induced ER stress response in MC3T3-E1 osteoblastic cells. a Cells were exposed to 400 μMH2O2 or 0.1 μMTg and 0, 25, 50, or 100 μg/mL PPHs for 6 h. Immunoblotting was performed using the indicated antibodies. b Cells were exposed to 400 μMH2O2 in the presence or absence of 100 μg/mL PPHs for 0, 2, 4, or 6 h. Immunoblotting was performed using the indicated antibodies. Blots are * representative of three independent experiments. p < 0.05 versus cells treated with H2O2 alone (n = 3). PPHs porcine placenta hydrolysates, Tg thapsigargin

apoptosis [31]. Thus, we evaluated the effect of PPHs on analyzed. H2O2 significantly increased the levels of caspase-12 activation. We found that caspase-12 activity caspase-12, caspase-3, and Bax in a time-dependent man- was increased significantly after H2O2 treatment, whereas ner; in contrast, treatment with PPHs markedly reduced treatment with PPHs markedly reduced this H2O2-in- the protein levels of caspase-12, caspase-3, and Bax produced increase in caspase-12 activity (Fig. 3c). Next, we tein in MC3T3-E1 cells (Fig. 3e). investigated caspase-3 activation. We found that H2O2 increased caspase-3 activation, whereas this increase was PPHs attenuate H2O2-mediated inhibition of ALP activity blocked by PPH treatment (Fig. 3d). Cells were exposed to To investigate the effect of PPHs on osteoblast differen- H2O2 in the absence or presence of PPHs for 2, 4, and 6 h, tiation, we investigated the mRNA level and activity of after which the levels of apoptosis-related proteins were ALP in MC3T3-E1 cells. Treatment with 400 μMH2O2 Lee et al. BMC Complementary and Alternative Medicine (2016) 16:291 Page 6 of 10

b a

c e

Fig. 3 PPHs protect against H2O2-induced apoptosis in MC3T3-E1 osteoblastic cells. a Apoptosis was detected by Hoechst dye staining and quantified based on nuclear condensation or fragmentation (right). Representative pictures are shown from three independent experiments. Images were taken at a magnification of 400×. Scale bar,20μm. b Cells were stained with FITC-conjugated Annexin V and PI, followed by flow

cytometric analysis. c Caspase-12 activity was analyzed in cells treated for 0, 12, 24, 36 or 48 h with 400 μMH2O2 in the presence or absence of 100 μg/mL PPHs. d Caspase-3 activity was measured in cells that were treated for 6 h with 400 μMH2O2 in the presence or absence of 100 μg/mL PPHs. e Cellsweretreatedfor0,2,4,or6hwith400μMH2O2 in the presence or absence of 100 μg/mL PPHs. Immunoblotting was performed using the indicated antibodies. Representative blots are shown from three independent experiments. Bottom panel, quantitative immunoblot data (n =4). * p < 0.05 versus cells treated with H2O2 alone. PPHs porcine placenta hydrolysates for 6 h significantly decreased the mRNA level of ALP. mRNA levels of Runx2 and osterix (Fig. 5b and c). How- However, treatment with PPHs markedly alleviated ever, PPHs markedly attenuated this H2O2-mediated H2O2-mediated downregulation of ALP. Similarly, treat- downregulation of Runx2 and osterix. ment for 6 h with 400 μMH2O2 markedly reduced ALP activity in cells. However, treatment with 100 μg/mL PPHs affect ROS release, NADPH oxidase, and SOD PPHs attenuated this effect of H2O2 (Fig. 4b). activity in MC3T3-E1 osteoblastic cells To evaluate the antioxidant effects of PPHs, we investi- PPHs attenuate H2O2-induced downregulation of gated the effect of PPHs on H2O2-mediated ROS pro- osteocalcin, Runx2, and osterix duction. We found that treatment with 400 μMH2O2 To investigate the effects of PPHs on bone formation for 2, 4, or 6 h markedly increased ROS production. proteins, we investigated the mRNA levels of osteocal- However, treatment with 100 μg/mL PPHs for 6 h mark- cin, Runx2 [32], and osterix [33]. Treatment for 6 h with edly attenuated this H2O2–mediated increase in intracel- 400 μMH2O2 markedly decreased the mRNA level of lular ROS (Fig. 6a). Next, we examined the effect of osteocalcin, whereas this change was markedly reversed PPHs on the activity of NADPH oxidase, an enzyme that by PPHs. Similarly, H2O2 markedly downregulated the generates ROS. As shown in Fig. 6b, PPHs reduced Lee et al. BMC Complementary and Alternative Medicine (2016) 16:291 Page 7 of 10

Discussion a In this study, we found that PPHs protect MC3T3-E1 cells against H2O2-induced cell death and suppression of differentiation. H2O2-induced oxidative stress is known to lead to pathological ER stress. We found that PPHs, presumably via their antioxidant effects, protected MC3T3-E1 osteoblastic cells against H2O2-induced ER stress and ROS production. Moreover, PPHs significantly reversed H2O2-mediated downregulation of osteocalcin and two related proteins involved in bone formation, namely osterix and Runx2. Reactive oxygen species (ROS) are generated by many environmental agents and have been implicated in the pathogenesis of various diseases, including osteoporosis [34]. Reduced bone formation by osteoblasts may cause osteoporosis to develop; moreover, the rate of osteoblast apoptosis regulates bone formation [1, 2, 35, 36]. ER stress has been implicated as an important factor in apoptosis due to various causes, including osteoblast apoptosis during the development of osteoporosis [11, 13, 14, 37]. The placenta has been studied for its potential to improve skin care, metabolic functions, and b strengthen immune function [23]. However, it has remained unclear whether the placenta can protect osteoblasts from H2O2-induced ER stress and apoptosis. Here we evaluated the effects of PPHs on H2O2-induced ER stress and apoptosis in osteoblasts. ROS production is known to decrease bone formation by osteoblastic cells by suppressing the differentiation of osteoblast pro- genitors and the calcification process [38]. Therefore, decreased bone formation is strongly associated with enhanced oxidative stress and significantly decreased plasma levels of antioxidants in elderly persons with osteoporosis [39, 40]. In this study, H2O2 induced cell death, ER stress, and caspase-mediated apoptosis (Figs. 1, 2 and 3). However, PPH treatment prevented caspase activation, thus reducing the amount of apoptosis in response to ER stress. ER stress was recently reported to Fig. 4 PPHs alleviate H2O2-mediated inhibition of ALP activity in be involved in the induction of apoptosis during osteo- MC3T3-E1 osteoblastic cells. a ALP mRNA levels in cells treated for porosis [11, 12, 14, 37]. ER stress is activated by the 6 h with 400 μMH2O2 in the presence or absence of 100 μg/mL PPHs were analyzed by RT-PCR. Representative bands are shown accumulation of unfolded proteins and alterations in cal- from three independent experiments. Quantitative analysis of RNA cium homeostasis [6–8]. Recent studies have also shown expression was also performed. b ALP activity levels were analyzed that ER stress-induced apoptosis of osteoblasts contrib- μ in cells treated with 400 MH2O2 in the presence or absence of utes to the development of osteoporosis [11–14, 37]. We μ * 100 g/mL PPHs (n = 3). p < 0.05 versus cells treated with H2O2 found that ER stress was decreased in MC3T3-E1 cells alone. PPHs porcine placenta hydrolysates (Fig. 2). However, treatment with PPHs increased osteoblast viability during periods of ER stress. Moreover, PPHs inhibited osteoblast apoptosis in response to ER H2O2-induced NADPH oxidase activity. These findings stress. These results demonstrate that PPHs can protect suggest that PPHs have antioxidant effects on osteo- osteoblasts from ER stress-induced apoptosis. blasts. Next, we investigated the effect of PPHs on SOD Caspases are the most prominent mediators of apop- activity. H2O2 treatment markedly inhibited SOD activ- tosis. However, it is not clear if activated caspase-12 or ity, whereas this effect was also alleviated by PPHs caspase-3 plays an important role in H2O2-induced (Fig. 6c). apoptosis [41]. In this study, we found that PPHs Lee et al. BMC Complementary and Alternative Medicine (2016) 16:291 Page 8 of 10

Fig. 5 PPHs reverse the effects of H2O2 on the mRNA expression of osteocalcin, Runx2, and osterix in MC3T3-E1 osteoblastic cells. Osteocalcin (a), Runx2 (b), and Osterix (c) expression levels in cells treated for 6 h with 400 μMH2O2 in the presence or absence of 100 μg/mL PPHs were analyzed by RT-PCR. Representative bands are shown from three independent experiments. Quantitative analysis of RNA expression was also performed. *p <0.05

versus cells treated with H2O2 alone. PPHs porcine placenta hydrolysates

reversed the H2O2-induced activation of caspase-12 and differentiation. We found that H2O2 markedly downreg- caspase-3 (Fig. 3). These results demonstrate that PPHs ulated ALP expression and that this effect was alleviated can protect osteoblasts from caspase-mediated, H2O2-in- by PPHs (Fig. 4). Our data thus indicate that PPHs duced apoptosis. stimulate osteoblast differentiation. Furthermore, we also H2O2-induced apoptosis is primary mediated by ROS analyzed the mRNA levels of Runx2, osterix, and osteo- production. We evaluated the protective effects of PPHs calcin. Osteocalcin is a major protein produced by osteo- and found that they decreased H2O2-induced ROS release blasts during bone formation. Osteocalcin expression is (Fig. 6). We also found that PPHs markedly inhibited regulated by Runx2 and osterix levels [32, 42]. There- NADPH oxidase activity. NADPH oxidase is important fore, we assessed the expression of osterix and Runx2 because it activates ROS-generating enzymes. PPHs also after treatment with PPHs and/or H2O2 (Fig. 5). We found alleviated H2O2-mediated inhibition of SOD activity. The that PPHs markedly reversed H2O2-mediated downregula- enhanced SOD activity observed upon treatment with tion of osterix and Runx2 expression. These findings sug- PPHs may result in the scavenging of excessive superox- gest that PPHs upregulate osteocalcin, osterix, and Runx2 ides derived from oxidative stress, thus ameliorating expression. H2O2-induced cell death. It was beyond the scope of the present study to iden- We also evaluated the effects of PPHs on osteoblastic tify the active component(s) in PPHs. However, recent differentiation. ALP is a primary marker of osteoblast studies have suggested that dietary arginine and lysine Lee et al. BMC Complementary and Alternative Medicine (2016) 16:291 Page 9 of 10

may play an important role in bone development, growth, a and modeling [43]. Moreover, arginine is thought to allevi- 250 ate metabolic disturbances in calcium absorption, growth, dentition, and decalcification [44]. PPHs are known to 200 contain high levels of arginine and other essential amino * acids. Thus, our findings provide evidence that PPHs may 150 have potential therapeutic value for treating bone metabolism disturbances in osteoblasts. 100 Conclusions 50 Our findings suggest that PPHs, presumably via their antioxidant effects, protect MC3T3-E1 osteoblasts against

ROS productionROS of (% control) 0 H2O2-induced ER stress by suppressing ROS production. Furthermore, PPHs regulate oxidative stress-induced osteoporosis. Cumulatively, our findings suggest that b PPHs may have potential therapeutic value for treating 200 bone formation disturbances.

Acknowledgments/Funding * This study was supported by a grant from Chonbuk National University 150 Hospital (to JHK) and a grant from the National Research Foundation (2015R1A2A1A13001849).

Availability of data and materials 100 All datasets supporting the conclusions of this article are included within the article. (% of control) of (%

Authors’ contributions 50

NADPH Oxidase activity HYL and JHK participated in the research design. HYL and HJC carried out the experiments, analyzed the data, and wrote the paper. SYP and JHK provided funding and supervised the study. All authors read and approved the final manuscript.

Competing interests c The authors declare that they have no competing interests. 120 Consent for publication Not applicable.

Ethics approval and consent to participate 80 * Not applicable.

Author details 1Department of Pharmacology and Institute of Drug Development, Chonbuk National University, Jeonju 560-182, Republic of Korea. 2CODEBIO CO., LTD, 40 Busong 1gil 62, Jiksan-eup, Seobuk-gu, Cheonan, Chungnam 331-815, Republic of Korea. 3Department of Obstetrics and Gynecology, Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju 560-182, Republic of Korea. SOD activitySOD of (% control) 0 Received: 5 January 2016 Accepted: 9 August 2016

References Fig. 6 PPHs regulate ROS release, NADPH oxidase, and SOD activity 1. Karsenty G, Wagner EF. Reaching a genetic and molecular understanding in MC3T3-E1 osteoblastic cells. a ROS production was analyzed in of skeletal development. Dev Cell. 2002;2(4):389–406. cells treated for 6 h with 400 μMHO in the presence or absence 2. Raisz LG. Pathogenesis of osteoporosis: concepts, conflicts, and prospects. 2 2 – of 100 μg/mL PPHs by the DCF-DA assay. NADPH oxidase (b) and J Clin Invest. 2005;115(12):3318 25. SOD activities (c) were assessed in cells treated for 6 h with 400 μM 3. Wu Y, Yang M, Fan J, Peng Y, Deng L, Ding Y, Yang R, Zhou J, Miao D, Fu Q. Deficiency of osteoblastic Arl6ip5 impaired osteoblast differentiation and H O in the presence or absence of 100 μg/mL PPHs. Representative 2 2 enhanced osteoclastogenesis via disturbance of ER calcium homeostasis bands are shown from three independent experiments. Quantitative and induction of ER stress-mediated apoptosis. Cell Death Dis. 2014;5:e1464. * analysis of RNA expression was also performed. p < 0.05 versus cells 4. Hock JM, Krishnan V, Onyia JE, Bidwell JP, Milas J, Stanislaus D. Osteoblast treated with H2O2 alone. PPHs, porcine placenta hydrolysates apoptosis and bone turnover. J Bone Miner Res Off J Am Soc Bone Miner Res. 2001;16(6):975–84. Lee et al. BMC Complementary and Alternative Medicine (2016) 16:291 Page 10 of 10

5. Downey PA, Siegel MI. Bone biology and the clinical implications for 30. Badham HJ, LeBrun DP, Rutter A, Winn LM. Transplacental benzene osteoporosis. Phys Ther. 2006;86(1):77–91. exposure increases tumor incidence in mouse offspring: possible role of 6. Schroder M. Endoplasmic reticulum stress responses. Cell Mol Life Sci. fetal benzene metabolism. Carcinogenesis. 2010;31(6):1142–8. 2008;65(6):862–94. 31. Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA, Yuan J. Caspase-12 7. Boyce M, Yuan J. Cellular response to endoplasmic reticulum stress: a mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by matter of life or death. Cell Death Differ. 2006;13(3):363–73. amyloid-beta. Nature. 2000;403(6765):98–103. 8. Hotamisligil GS. Endoplasmic reticulum stress and the inflammatory basis of 32. Gersbach CA, Byers BA, Pavlath GK, Garcia AJ. Runx2/Cbfa1 stimulates metabolic disease. Cell. 2010;140(6):900–17. transdifferentiation of primary skeletal myoblasts into a mineralizing 9. Hasnain SZ, Lourie R, Das I, Chen AC, McGuckin MA. The interplay between osteoblastic phenotype. Exp Cell Res. 2004;300(2):406–17. endoplasmic reticulum stress and inflammation. Immunol Cell Biol. 2012; 33. Komori T. Regulation of osteoblast differentiation by transcription factors. 90(3):260–70. J Cell Biochem. 2006;99(5):1233–9. 10. Yamada H, Nakajima T, Domon H, Honda T, Yamazaki K. Endoplasmic 34. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. reticulum stress response and bone loss in experimental periodontitis in Nature. 2000;408(6809):239–47. mice. J Periodontal Res. 2015;50(4):500–8. 35. Rodan GA, Martin TJ. Therapeutic approaches to bone diseases. Science. 11. Park SJ, Kim KJ, Kim WU, Oh IH, Cho CS. Involvement of endoplasmic 2000;289(5484):1508–14. reticulum stress in homocysteine-induced apoptosis of osteoblastic cells. 36. Weinstein RS, Manolagas SC. Apoptosis and osteoporosis. Am J Med. J Bone Miner Metab. 2012;30(4):474–84. 2000;108(2):153–64. 12. Tang CH, Chiu YC, Huang CF, Chen YW, Chen PC. Arsenic induces cell 37. Hino S, Kondo S, Yoshinaga K, Saito A, Murakami T, Kanemoto S, Sekiya H, apoptosis in cultured osteoblasts through endoplasmic reticulum stress. Chihara K, Aikawa Y, Hara H, et al. Regulation of ER molecular chaperone Toxicol Appl Pharmacol. 2009;241(2):173–81. prevents bone loss in a murine model for osteoporosis. J Bone Miner – 13. Lisse TS, Thiele F, Fuchs H, Hans W, Przemeck GK, Abe K, Rathkolb B, Metab. 2010;28(2):131 8. Quintanilla-Martinez L, Hoelzlwimmer G, Helfrich M, et al. ER stress-mediated 38. Hosoya S, Suzuki H, Yamamoto M, Kobayashi K, Abiko Y. Alkaline apoptosis in a new mouse model of osteogenesis imperfecta. PLoS Genet. phosphatase and type I collagen gene expressions were reduced by 2008;4(2):e7. hydroxyl radical-treated fibronectin substratum. Mol Genet Metab. – 14. He L, Lee J, Jang JH, Sakchaisri K, Hwang J, Cha-Molstad HJ, Kim KA, Ryoo IJ, 1998;65(1):31 4. Lee HG, Kim SO, et al. Osteoporosis regulation by salubrinal through 39. Basu S, Michaelsson K, Olofsson H, Johansson S, Melhus H. Association eIF2alpha mediated differentiation of osteoclast and osteoblast. Cell Signal. between oxidative stress and bone mineral density. Biochem Biophys Res – 2013;25(2):552–60. Commun. 2001;288(1):275 9. 15. Raggatt LJ, Partridge NC. Cellular and molecular mechanisms of bone 40. Maggio D, Barabani M, Pierandrei M, Polidori MC, Catani M, Mecocci P, remodeling. J Biol Chem. 2010;285(33):25103–8. Senin U, Pacifici R, Cherubini A. Marked decrease in plasma antioxidants in 16. Raisz LG. Physiology and pathophysiology of bone remodeling. Clin Chem. aged osteoporotic women: results of a cross-sectional study. J Clin – 1999;45(8 Pt 2):1353–8. Endocrinol Metab. 2003;88(4):1523 7. 17. Aggarwal BB. Targeting inflammation-induced obesity and metabolic 41. Singh M, Sharma H, Singh N. Hydrogen peroxide induces apoptosis in – diseases by curcumin and other nutraceuticals. Annu Rev Nutr. 2010;30:173–99. HeLa cells through mitochondrial pathway. Mitochondrion. 2007;7(6):367 73. 42. Hassan MQ, Javed A, Morasso MI, Karlin J, Montecino M, van Wijnen AJ, 18. Bhandari P, Song M, Dorn 2nd GW. Dissociation of mitochondrial from Stein GS, Stein JL, Lian JB. Dlx3 transcriptional regulation of osteoblast sarcoplasmic reticular stress in Drosophila cardiomyopathy induced by differentiation: temporal recruitment of Msx2, Dlx3, and Dlx5 molecularly distinct mitochondrial fusion defects. J Mol Cell Cardiol. homeodomain proteins to chromatin of the osteocalcin gene. Mol Cell Biol. 2015;80:71–80. 2004;24(20):9248–61. 19. Cheng AC, Tsai ML, Liu CM, Lee MF, Nagabhushanam K, Ho CT, Pan MH. 43. Sahin K, Onderci M, Sahin N, Balci TA, Gursu MF, Juturu V, Kucuk O. Garcinol inhibits cell growth in hepatocellular carcinoma Hep3B cells through Dietary arginine silicate inositol complex improves bone mineralization in induction of ROS-dependent apoptosis. Food Funct. 2010;1(3):301–7. quail. Poult Sci. 2006;85(3):486–92. 20. Guo YS, Sun Z, Ma J, Cui W, Gao B, Zhang HY, Han YH, Hu HM, Wang L, 44. Fiore CE, Pennisi P, Cutuli VM, Prato A, Messina R, Clementi G. L-arginine Fan J, et al. 17beta-Estradiol inhibits ER stress-induced apoptosis through prevents bone loss and bone collagen breakdown in cyclosporin A-treated promotion of TFII-I-dependent Grp78 induction in osteoblasts. Lab Invest. rats. Eur J Pharmacol. 2000;408(3):323–6. 2014;94(8):906–16. 21. Hamamura K, Yokota H. Stress to endoplasmic reticulum of mouse osteoblasts induces apoptosis and transcriptional activation for bone remodeling. FEBS Lett. 2007;581(9):1769–74. 22. Xiong Z, Jiang R, Zhang P, Han X, Guo FJ. Transmission of ER stress response by ATF6 promotes endochondral bone growth. J Orthop Surg Res. 2015;10:141. 23. Togashi S, Takahashi N, Iwama M, Watanabe S, Tamagawa K, Fukui T. Antioxidative collagen-derived peptides in human-placenta extract. Placenta. 2002;23(6):497–502. 24. Yamasaki M, Hasegawa S, Takahashi H, Kobayashi Y, Sakai C, Ashizawa Y, Asai Y, Kanzaki M, Fukui T. Placental extracts induce the expression of antioxidant enzyme genes and suppress melanogenesis in B16 melanoma Submit your next manuscript to BioMed Central cells. Nat Prod Res. 2015;29(22):2103–6. 25. Hong JW, Lee WJ, Hahn SB, Kim BJ, Lew DH. The effect of human placenta and we will help you at every step: extract in a wound healing model. Ann Plast Surg. 2010;65(1):96–100. 26. Nath S, Bhattacharyya D. Cell adhesion by aqueous extract of human • We accept pre-submission inquiries placenta used as wound healer. Indian J Exp Biol. 2007;45(8):732–8. • Our selector tool helps you to ﬁnd the most relevant journal 27. Fang XP, Xia WS, Sheng QH, Wang YL. Purification and characterization of • We provide round the clock customer support an immunomodulatory Peptide from bovine placenta water-soluble extract. Prep Biochem Biotechnol. 2007;37(3):173–84. • Convenient online submission 28. Lee KH, Park HJ, Seo HG, Kim JH, Lim GS, Lee WY, Kim NH, Kim JH, Lee JH, • Thorough peer review Jung HS, et al. Immune modulation effect of porcine placenta extracts in • Inclusion in PubMed and all major indexing services weaned the pig. J Anim Sci. 2013;91(5):2405–13. • 29. Zayzafoon M. Calcium/calmodulin signaling controls osteoblast growth and Maximum visibility for your research differentiation. J Cell Biochem. 2006;97(1):56–70. Submit your manuscript at www.biomedcentral.com/submit